Our objective is to develop a commercially viable influenza diagnostic for rapid and simultaneous screening of clinical samples for influenza A and B type and important subtypes. The advantages of the proposed approach over existing polymerase chain reaction (PCR) methods for influenza viruses stem from the use of a single gene segment for identification of both type and subtype. The target for the proposed work, the matrix gene segment, is known to be robustly amplified and more conserved than the traditional hemagglutinin gene (HA) target. The initial commercial niche for the developed product will be state and local public health laboratories. While state health labs currently receive clinical samples for typing and subtyping, the cost of subtyping by the "gold standard" of viral isolation and hemagglutination inhibition test is prohibitive. Currently, most health labs rely on a fluorescence-based immunoassay to screen for H3 or H1 viruses, with no confirmation of the neuraminidase subtype and no current capabilities for emerging viruses. Those labs that utilize real-time reverse transcription PCR (RRT-PCR) assays must rely on the mutation- susceptible HA gene for partial subtyping and must conduct individual tests for each HA subtype. The proposed product would serve as a means to improve and broaden surveillance efforts at state and local levels in the US, as well as in regional labs worldwide, by providing a rapid and cost-effective means to simultaneously screen for type (A and B) and certain subtypes, specifically, current human-adapted influenza viruses (A/H3N2 and A/H1N1) and A/H5N1. This new surveillance tool would be used in place of existing immunoassays and singleplex HA targeted RT-PCR based assays but is not designed to replace viral isolation methods and sequencing, which are necessary for a more complete understanding of influenza viruses. Specific Aim 1 will capitalize on the recent discovery that the matrix gene segment of influenza's viral genome can provide both type and subtype information for influenza viruses. The hypothesis to be tested is that a small set of M gene segment specific primers pairs (5) for RT-PCR can be designed to selectively detect A/H3N2, A/H1N1, A/H5N1, and B viruses. Specific Aim 2 will focus on systematic optimization of multiplex conditions for RT-PCR. In Specific Aim 3, promising primer sets that satisfy the criteria for success with initial samples will be validated in a blind study of 300+ patient samples acquired over at least two flu seasons by a variety of sampling methods, including nasal swab, nasal wash, and nasopharyngeal aspiration. In Phase 2 we will engineer a system for automated sample handling, including extraction and RT-PCR amplification, followed by rapid separation and detection of PCR products by a fast chromatographic method. Post-PCR separation and detection is anticipated to provide superior accuracy and sensitivity relative to multiplex RRT- PCR with no added time to the overall assay. The motivation for the proposed work is the tremendous impact influenza viruses have on human and animal health and the need for rapid, inexpensive tools for strain surveillance. The intent is to provide state and local public health laboratories with the ability to affordably and rapidly screen patient samples for influenza type and subtype using the highly reliable and conserved matrix gene segment as the identification target.